Nanopore Based Device for Cutting Long DNA Molecules into Fragments

ABSTRACT

Apparatus, system, and method are provided for cutting a linear charged polymer inside a nanopore. A first voltage is applied to create an electric field in a first direction. A second voltage is applied to create an electric field in a second direction, and the first direction is opposite to the second direction. When the electric field in the first direction and the electric field in the second direction are applied to a linear charged polymer inside a nanopore, the linear charged polymer is cut at a location with predetermined accuracy.

BACKGROUND

Exemplary embodiments relate to nanodevices, and more specifically, to ananopore device for cutting polymers.

Recently, there has been growing interest in applying nanopores assensors for rapid analysis of biomolecules (DNA, RNA, protein, etc).Special emphasis has been given to applications of nanopores for DNAsequencing, as this technology holds the promise to reduce the cost ofsequencing below $1000/human genome. An issue in these applications isthe control of the translocation of DNA through the nanopore.

Nanopore sequencing is a method for determining the order in whichnucleotides occur on a strand of DNA. A nanopore is simply a small holeof the order of several nanometers in internal diameter. The theorybehind nanopore sequencing has to do with what occurs when the nanoporeis immersed in a conducting fluid and an electric potential (voltage) isapplied across it: under these conditions a slight electric current dueto conduction of ions through the nanopore can be measured, and theamount of current is very sensitive to the size and shape of thenanopore. If single bases or strands of DNA pass (or part of the DNAmolecule passes) through the nanopore, this can create a change in themagnitude of the current through the nanopore. Other electrical oroptical sensors can also be put around the nanopore so that DNA basescan be differentiated while the DNA passes through the nanopore.

DNA could be driven through the nanopore by using various methods. Forexample, an electric field might attract the DNA towards the nanopore,and it might eventually pass through it. Also, enzymes attached to thenanopore might guide DNA towards the nanopore. The scale of the nanoporemeans that the DNA may be forced through the hole as a long string, onebase at a time, rather like thread through the eye of a needle.

BRIEF SUMMARY

According to one exemplary embodiment, a method is provided for cuttinga linear charged polymer inside a nanopore. A first voltage is appliedto create an electric field in a first direction. A second voltage isapplied to create an electric field in a second direction, and the firstdirection is opposite to the second direction. When the electric fieldin the first direction and the electric field in the second directionare applied to a linear charged polymer inside a nanopore, the linearcharged polymer is caused to be cut at a location with a specifiedprecision.

Other systems, methods, apparatus, design structures, and/or computerprogram products according to embodiments will be or become apparent toone with skill in the art upon review of the following drawings anddetailed description. It is intended that all such additional systems,methods, apparatus, design structures, and/or computer program productsbe included within this description, be within the scope of theexemplary embodiments, and be protected by the accompanying claims. Fora better understanding of the features, refer to the description and tothe drawings.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The subject matter which is regarded as the invention is particularlypointed out and distinctly claimed in the claims at the conclusion ofthe specification. The forgoing and other features are apparent from thefollowing detailed description taken in conjunction with theaccompanying drawings in which:

FIGS. 1 and 2 illustrate a schematic of a nanopore device in accordancewith exemplary embodiments.

FIG. 3 illustrates a method of cutting a polymer in accordance withexemplary embodiments.

FIG. 4 shows a block diagram of an exemplary design flow used forexample, in semiconductor IC logic design, simulation, test, layout, andmanufacture of exemplary embodiments.

FIG. 5 illustrates a library of DNA fragments (or any linear chargedpolymer) in accordance with exemplary embodiments.

DETAILED DESCRIPTION

Exemplary embodiments provide a method and mechanism to perform DNArestriction, which is a process whereby a long DNA molecule is cut intoshorter fragments.

Unlike exemplary embodiments, conventional fragmentation methods can bephysical (e.g., nebulization, sonication and hydrodynamic shearing) orenzymatic. Enzymatic restriction is achieved using an enzyme thatrecognizes and cuts DNA molecules at specific sequence patterns(restriction sites). DNA restriction is routinely used in preparation ofsamples. Many applications such as chip-on-chip and DNA sequencing, fromthe original Sanger sequencing to most large-scale DNA sequencingstrategies, require the fragmentation of the DNA to be sequenced forsub-cloning and further processing. In these applications, a long DNAmolecule containing tens of thousands of bases must be cut into shorterfragments with a typical length of 100 to 1000 bases. Both in thephysical as well as enzymatic fragmentation methods DNA fragments have avariable length whose distribution depends on the method used forfragmentation. In the sequencing applications, producing many short DNAfragments would increase the cost of sequencing, while producing longDNA fragments could potentially reduce the accuracy of sequencing.

In order to optimize the outcome in these applications, it might bedesirable to produce a library of DNA fragments with a prescribed lengthutilizing the DNA transistor described in the patent application“Systems and methods for controlling the position of a charged polymerinside a nanopore”, U.S. patent publication number 2008/0187915 which isherein incorporated by reference. The DNA transistor is a device thatallows for the controlled translocation of DNA molecules though a solidnanopore at a single-nucleotide resolution. A DNA transistor contains ametal-dielectric-metal-dielectric-metal multi-layered solid membrane anda few nanometer sized pore drilled through such solid membrane. Whenproperly chosen voltages are applied to the metal layers, DNA can beeffectively trapped within the DNA transistor. The voltage applied tothe metal layers produces an electric field that effectively traps DNAmolecule. Switching on and off such voltages in the presence of abiasing field corresponds to stopping and moving states of DNA,respectively. Since the DNA molecule translocates at a rate of onenucleotide per cycle of on-off voltages on the electrodes of the DNAtransistor, it is possible to count the number of bases that translocatethrough the pore.

In accordance with exemplary embodiments, after a desired number of DNAbases have translocated, the DNA molecule can be stopped by applying thetrapping potential for a period of time. Under this condition, DNA canbe electrically stretched and broken into two pieces at a pre-determinedlocation within the DNA transistor using the features described hereinin accordance with exemplary embodiments. After cutting the DNAaccording to exemplary embodiments, the short DNA fragment can beelectrically driven through the nanopore to exit the membrane, while therest of the long DNA fragment continues moving forward base by base tostart a new cutting cycle.

In exemplary embodiments, it is possible to cut sequences at a desiredsequence pattern. In order to do this, it should be noted that inprinciple it is possible to identify the sequence of nucleotides thatwent through the pore. This can be done by detecting voltage signalssuch as capacitance changes or tunneling current due to the presence ofone or more nucleotides in the proximity of strategically locatedsensors within the nanopore during the “stop” phase of thetranslocation. After a desired DNA sequence pattern is detected, the DNAmolecule can be stopped by applying the trapping potential for a longtime. Under this condition, DNA can be electrically stretched and brokeninto two pieces at a pre-determined location within the DNA transistorusing the features described herein in accordance with exemplaryembodiments. After cutting the DNA, the DNA fragment closer to the exitof the pore can be electrically driven through the nanopore to exit themembrane.

For example, exemplary embodiments disclose a device 100 (shown inFIG. 1) that cuts a DNA molecule (or any linear charged polymer) at aprescribed length and/or at a prescribed sequence pattern by means ofthe application of electrical voltages within the DNA transistor, thusallowing the device to act as “DNA electric scissors”. Note that cuttingDNA with DNA electric scissors does not require complicated samplepreparations or expensive enzymes. The cost to electrically “cut” longDNA into small fragments of controlled size and/or at predeterminedsequences is expected to be substantially lower than the cost involvedin traditional methods. Note also that while some restriction enzymescut sequences at specific DNA sequences in single stranded DNA, therestriction enzymes seem to do so at transient double stranded regionsformed by hybridization of complementary sequences within the samesingle stranded molecule. In this respect, the DNA scissors of exemplaryembodiments in the mode in which they cut DNA molecules at specificsequence patterns do so in single stranded DNA molecules without theneed of the complementary strand within the same molecule in accordancewith exemplary embodiments.

Now turning to FIG. 1 in detail, FIG. 1 illustrates the cross-section ofa device 100 having a long DNA in a DNA transistor in accordance withexemplary embodiments. It is noted that the device 100 is not drawn toscale but is drawn to illustrate various features as understood by oneskilled in the art. A membrane 120, which is made of films 101, 102,103, 104, and 105, partitions a reservoir 106 into two parts.

A nanometer size hole 107, referred to as a nanopore, is made throughthe membrane 120. The reservoir 106 and the hole 107 are then filledwith ionic buffer 108. The ionic buffer 108 may be a salt and watersolution.

The reservoir 106 may be an insulated container that is configured tohold the solution of the conductive ionic buffer 108. The membrane parts101, 103, and 105 are made of electrically conducting materials, whilemembrane part 102 and 104 are made of insulating materials. The voltagebias 109 is applied between membrane parts 101 and 103 with thedirection defined by the battery sign. Likewise, the voltage bias 110 isapplied between membrane parts 103 and 105 with the direction defined bythe battery sign. A polymer, such as a DNA molecule 111, with discretecharges 112 can be driven into the nanopore 107 by applying a voltagebias 113 through two electrodes 114 and 115, which are immersed in thetwo parts of the ionic buffers 108.

The translocation of the DNA molecule 111 can be precisely controlled ata single-base resolution by applying the proper voltages 109 and 110 onand off. By the voltage biases 109 and 110, electric fields 122 and 124(in the regions of 102 and 104) are produced to interact with thediscrete charges 112 of the DNA molecule 111 (in the regions of 102 and104). When the DNA molecule 111 moves downward, there is an imbalance offorces on the DNA molecule 111 in the regions 102 and 104, and the netforce on the DNA molecule 111 is upward. Conversely, when the DNAmolecule 111 moves upward the electric fields 122 and 124 create animbalance of forces on the DNA molecule 111, and the net force on theDNA molecule 111 is downward. Thus, the voltages 109 and 110 will trapthe DNA molecule 111 at the minimal of a trapping potential well whenthe electric fields 122 and 124 cause the forces on the DNA molecule 111to be balanced. For example, the voltages 109 and 110 can keep the DNAmolecule 111 in place like a steady state and cause a tug of war betweenthe forces on the DNA molecule 111 so that the DNA molecule 111 remainsstill in the nanopore 107. For further information regarding controllinga DNA molecule, reference is made to “Nanopore in metal-dielectricsandwich for DNA position control” in Applied Physics Letters 91, 153103(2007) by Stas Polonsky, Steve Rossnagel, and Gustavo Stolovitzky, whichis herein incorporated by reference.

The thicknesses of the insulating layers 102 and 104 and the conductivelayer 103 should be properly chosen to be commensurate with the spacingbetween neighboring phosphate groups in the DNA molecule 111. As oneexample in exemplary embodiments, a rule of thumb that may be utilized(but not an essential configuration) is one in which the thickness ofeach insulating layer 102 and 104 is equal to (n+0.5)d and the thicknessof the middle conductive layer 103 is equal to md, where m and n aresmall integers (e.g., between 2 and 10) and where d is the spacingbetween neighboring phosphate groups in the DNA molecule 111.Accordingly, the thickness of the insulating layers 102 and 104 and thethickness of the middle conductive layer 103 may be (but are not limitedto) a function of the spacing d between the phosphate groups in the DNAmolecule 111 being cut according to exemplary embodiments.

It is understood by one skilled in the art that double stranded DNAconsists of two long polymers of simple units called nucleotides, withbackbones made of sugars and phosphate groups joined by ester bonds.These two strands run in opposite directions to each other and aretherefore anti-parallel. Attached to each sugar is one of four types ofmolecules called bases. It is the sequence of these four bases along thebackbone that encodes biological information.

In accordance with exemplary embodiments, there are three operationalstages during a DNA-cutting cycle. As shown in FIG. 1, the electricfields 122 and 124 in the insulating layers 102 and 104 have the samestrength but opposite directions. Note that the representation of theelectric fields 122 and 124 is shown on the right side of FIGS. 1 and 2so as not to obscure the drawings, but it is understood that theelectric fields 122 and 124 are across the films 102 and 104 to interactwith the DNA molecule 111. Each time the voltages 109 and 110 switchfrom +V to −V in the presence of a biasing electric voltage 113 from thetop to the bottom (trans to cis) of the compartment of the reservoir106, the DNA strand 111 is driven from the top compartment by half aspacing between neighboring phosphate groups due to the swapping of themaximum and minimum of the electrical trapping energy of the DNAmolecule 111. If this switching (from +V to −V of the voltages 109 and110) is done periodically, the translocation will be at a rate of onebase per period. After the DNA molecule 111 advances a prescribed length(and/or if the DNA molecule 111 is simultaneously being sequenced untilthe right sequence is identified), the cycling stops (from +V to −V ofthe voltages 109 and 110), and the voltages 109 and 110 are set tomaintain, e.g., a voltage having a magnitude of V. Under thisconfiguration the DNA molecule 111 is trapped in the nanopore 107. Inexemplary embodiments, the typical but not limiting value of V fortrapping DNA is about 1 or 2 volts.

With reference to FIG. 2, the electric fields 122 and 124 in theinsulating layers 102 and 104 respectively point downwards and upwards,and the DNA molecule 111 is slightly stretched by the electric fields122 and 124 in exemplary embodiments. The electric fields 122 and 124pointing toward each other may be increased (by increasing respectivevoltages 109 and 110), and the electric fields 122 and 124 pointingtoward each other exert forces on the DNA fragments (in two electricfield regions) in opposite directions, pulling the two DNA fragmentsaway from each other. In other words, although the electric fields 122and 124 point toward each other, the combination of forces interactingon the DNA molecule 111 stretch the DNA molecule 111 to cause the cut,which is illustrated at location 120. For example, one electric fieldregion is the area around the insulating layer 102 and the otherelectric field region is the area around the insulating layer 104; theDNA fragments (or portions) of the DNA molecule 111 that are in thoseelectric field regions have forces that cause the DNA fragment (portion)in one electric field region to pull away from the DNA fragment(portion) in the other electric field region.

Further, in the DNA-cutting mode, an additional voltage pulse V′=V₀ isadded to voltages 109 and 110 for a duration δ in accordance withexemplary embodiments. When V₀>4 volts, the stretching force on the DNAmolecule 111 is equal to F=e(V+V₀)/d>1 nN, which is strong enough tobreak the DNA backbone of the DNA molecule 111 near electrode 103(identified by the circle 120). In the equation above, e is one electroncharge (corresponding to the charge of one nucleotide of the DNAmolecule 111), d is the spacing between the negative charges 112 (of thephosphate groups) of the DNA molecule 111, V is the trapping voltage,and V₀ is the cutting voltage needed to cut the DNA molecule 111 asshown in FIG. 2. Also, nN is a nanonewton. Note that the voltage pulseis only applied for a time δ in order to avoid possible electrochemicalreactions when applying a high voltage on the electrodes 114 and 115. Inaddition, although V₀>4 is provided as an example voltage to cut the DNAmolecule 111, the value of V₀ is not meant to be limiting and othervalues for V₀ may be utilized in accordance the present disclosure asunderstood by one skilled in the art.

Additionally, in exemplary embodiments, the location 120 of the break tocut the DNA molecule 111 can be tuned by making the thickness of themetal layer 103 smaller. Since the DNA molecule 111 is cut in thelocation 120 at the metal layer (film) 103, the metal layer 103 can bemade smaller to provide a more precise cut of the DNA molecule 111.

Now turning to FIG. 5, FIG. 5 illustrates a library 500 of DNA fragments(or any linear charged polymer) in accordance with exemplaryembodiments. The library 500 is a collection of DNA fragments that isphysically stored in a solution in containers 502, and the containers502 may be any container capable of storing DNA as understood by oneskilled in the art. For example, the DNA fragments may be stored in atest tube.

In accordance with exemplary embodiments, the device 100 can cut the DNAmolecule 111 into many different DNA fragments of different lengths, andthe DNA fragments can be organized and stored in the library 500. TheDNA fragments can be cut into different lengths and stored in thecontainer 502.

For example, a user of the device 100 may cut the DNA molecule 111 intothe desired sizes (i.e., lengths of the DNA fragments) and monitor thecutting process. The user of the device 100 may cut the DNA fragmentsinto a proportion of different lengths as desired for storage in thecontainer 502. For example, in each container 502, for each DNA fragmentof length 100, there may be 2 DNA fragments of length 135, 7 DNAfragments of length 250, and so on. Accordingly, utilizing the device100, the user can cut and store (in each container 502 of the library500) DNA fragments in an arbitrary desired proportion, and/or with anydesired distribution of sizes.

FIG. 3 illustrates a method 300 for cutting linear charged polymersinside a nanopore in accordance with exemplary embodiments.

A first voltage (such as the voltage 109) is applied to create anelectric field (such as the electric field 122) in a first direction atoperation 302.

A second voltage (such as the voltage 110) is applied to create anelectric field (such as the electric field 124) in a second direction atoperation 304. The first direction is opposite to the second direction.

When the electric field in the first direction and the electric field inthe second direction are applied to a linear charged polymer (such asthe DNA molecule 111) inside the nanopore 107, the electric fields causethe linear charged polymer 111 to be cut at a precise location (such asthe location 120) at operation 306.

Further, a third voltage (such as the voltage 113) is applied to be abiasing voltage that moves the linear charged polymer 111 inside thenanopore 107. Cutting the linear charged polymer 111 at the preciselocation 120 comprises stretching the linear charged polymer 111 untilthe linear charged polymer 111 breaks at the precise location 120. Whenthe electric field 122 in the first direction and the electric field 124in the second direction are applied to the linear charged polymer 111inside the nanopore 107 to cause the linear charged polymer 111 to becut at the precise location 120, the linear charged polymer 111 is cutinto pieces at every predetermined length. The predetermined length tocut the linear charged polymer 111 may be, e.g., every X amount ofunits. For example, the linear charged polymer 111 may be cut within apredetermined accuracy, such as every 100 nucleotides plus or minus 5nucleotides. That is, the linear charged polymer 111 may be cut everycertain number of nucleotides within a certain accuracy as desired. TheEach unit in the DNA molecule 111 may correspond to each charge 112 inthe phosphate group, and the DNA molecule 111 can be cut every X numberof charges 112. Also, the linear charged polymer 111 can be cut at apredetermined distance based on a particular pattern of monomers.

The linear charged polymer 111 is not meant to be limiting and maycomprise a cationic polysaccharide, deoxyribonucleic acid (DNA),ribonucleic acid (RNA), and/or other polymers. In accordance withexemplary embodiments, both the first voltage and the second voltage areapplied such that the electric field in the first direction and theelectric field in the second direction are strong enough to break thelinear charged polymer 111 at the precise location 120.

It is understood by one skilled in the art that a polymer is a largemolecule (macromolecule) composed of repeating structural unitstypically connected by covalent chemical bonds and in exemplaryembodiments, the polymer can be cut every X repeating units. Due to theextraordinary range of properties accessible in polymeric materials,they have come to play an essential and ubiquitous role in everydaylife, from plastics and elastomers on the one hand to naturalbiopolymers such as DNA and proteins that are essential for life on theother. A simple example is polyethylene, whose repeating unit is basedon ethylene monomer. Most commonly, as in this example, the continuouslylinked backbone of a polymer used for the preparation of plasticsconsists mainly of carbon atoms. However, the backbone of DNA is in factbased on a phosphodiester bond, and repeating units of polysaccharides(e.g. cellulose) are joined together by glycosidic bonds via oxygenatoms.

FIG. 4 shows a block diagram of an exemplary design flow 400 used forexample, in semiconductor IC logic design, simulation, test, layout, andmanufacture. Design flow 400 includes processes, machines and/ormechanisms for processing design structures or devices to generatelogically or otherwise functionally equivalent representations of thedesign structures and/or devices described above and shown in FIGS. 1and 2. The design structures processed and/or generated by design flow400 may be encoded on machine-readable transmission or storage media toinclude data and/or instructions that when executed or otherwiseprocessed on a data processing system generate a logically,structurally, mechanically, or otherwise functionally equivalentrepresentation of hardware components, circuits, devices, or systems.Machines include, but are not limited to, any machine used in an ICdesign process, such as designing, manufacturing, or simulating acircuit, component, device, or system. For example, machines mayinclude: lithography machines, machines and/or equipment for generatingmasks (e.g. e-beam writers), computers or equipment for simulatingdesign structures, any apparatus used in the manufacturing or testprocess, or any machines for programming functionally equivalentrepresentations of the design structures into any medium (e.g. a machinefor programming a programmable gate array).

Design flow 400 may vary depending on the type of representation beingdesigned. For example, a design flow 400 for building an applicationspecific IC (ASIC) may differ from a design flow 400 for designing astandard component or from a design flow 400 for instantiating thedesign into a programmable array, for example a programmable gate array(PGA) or a field programmable gate array (FPGA) offered by Altera® Inc.or Xilinx® Inc.

FIG. 4 illustrates multiple such design structures including an inputdesign structure 420 that is preferably processed by a design process410. Design structure 420 may be a logical simulation design structuregenerated and processed by design process 410 to produce a logicallyequivalent functional representation of a hardware device. Designstructure 420 may also or alternatively comprise data and/or programinstructions that when processed by design process 410, generate afunctional representation of the physical structure of a hardwaredevice. Whether representing functional and/or structural designfeatures, design structure 420 may be generated using electroniccomputer-aided design (ECAD) such as implemented by a coredeveloper/designer. When encoded on a machine-readable datatransmission, gate array, or storage medium, design structure 420 may beaccessed and processed by one or more hardware and/or software moduleswithin design process 410 to simulate or otherwise functionallyrepresent an electronic component, circuit, electronic or logic module,apparatus, device, or system such as those shown in FIGS. 1 and 2. Assuch, design structure 420 may comprise files or other data structuresincluding human and/or machine-readable source code, compiledstructures, and computer-executable code structures that when processedby a design or simulation data processing system, functionally simulateor otherwise represent circuits or other levels of hardware logicdesign. Such data structures may include hardware-description language(HDL) design entities or other data structures conforming to and/orcompatible with lower-level HDL design languages such as Verilog andVHDL, and/or higher level design languages such as C or C++.

Design process 410 preferably employs and incorporates hardware and/orsoftware modules for synthesizing, translating, or otherwise processinga design/simulation functional equivalent of the components, circuits,devices, or logic structures shown in FIGS. 1 and 2 to generate anetlist 480 which may contain design structures such as design structure420. Netlist 480 may comprise, for example, compiled or otherwiseprocessed data structures representing a list of wires, discretecomponents, logic gates, control circuits, I/O devices, models, etc.that describes the connections to other elements and circuits in anintegrated circuit design. Netlist 480 may be synthesized using aniterative process in which netlist 480 is resynthesized one or moretimes depending on design specifications and parameters for the device.As with other design structure types described herein, netlist 480 maybe recorded on a machine-readable data storage medium or programmed intoa programmable gate array. The medium may be a non-volatile storagemedium such as a magnetic or optical disk drive, a programmable gatearray, a compact flash, or other flash memory. Additionally, or in thealternative, the medium may be a system or cache memory, buffer space,or electrically or optically conductive devices and materials on whichdata packets may be transmitted and intermediately stored via theInternet, or other networking suitable means.

Design process 410 may include hardware and software modules forprocessing a variety of input data structure types including netlist480. Such data structure types may reside, for example, within libraryelements 430 and include a set of commonly used elements, circuits, anddevices, including models, layouts, and symbolic representations, for agiven manufacturing technology (e.g., different technology nodes, 32 nm,45 nm, 90 nm, etc.). The data structure types may further include designspecifications 440, characterization data 450, verification data 460,design rules 470, and test data files 485 which may include input testpatterns, output test results, and other testing information. Designprocess 410 may further include, for example, standard mechanical designprocesses such as stress analysis, thermal analysis, mechanical eventsimulation, process simulation for operations such as casting, molding,and die press forming, etc. One of ordinary skill in the art ofmechanical design can appreciate the extent of possible mechanicaldesign tools and applications used in design process 410 withoutdeviating from the scope and spirit of the invention. Design process 410may also include modules for performing standard circuit designprocesses such as timing analysis, verification, design rule checking,place and route operations, etc. Design process 410 employs andincorporates logic and physical design tools such as HDL compilers andsimulation model build tools to process design structure 420 togetherwith some or all of the depicted supporting data structures along withany additional mechanical design or data (if applicable), to generate asecond design structure 490. Design structure 490 resides on a storagemedium or programmable gate array in a data format used for the exchangeof data of mechanical devices and structures (e.g. information stored ina IGES, DXF, Parasolid XT, JT, DRG, or any other suitable format forstoring or rendering such mechanical design structures). Similar todesign structure 420, design structure 990 preferably comprises one ormore files, data structures, or other computer-encoded data orinstructions that reside on transmission or data storage media and thatwhen processed by an ECAD system generate a logically or otherwisefunctionally equivalent form of one or more of the embodiments of theinvention shown in FIGS. 1 and 2. In one embodiment, design structure490 may comprise a compiled, executable HDL simulation model thatfunctionally simulates the devices shown in FIGS. 1 and 2.

Design structure 490 may also employ a data format used for the exchangeof layout data of integrated circuits and/or symbolic data format (e.g.information stored in a GDSII (GDS2), GL1, OASIS, map files, or anyother suitable format for storing such design data structures). Designstructure 490 may comprise information such as, for example, symbolicdata, map files, test data files, design content files, manufacturingdata, layout parameters, wires, levels of metal, vias, shapes, data forrouting through the manufacturing line, and any other data required by amanufacturer or other designer/developer to produce a device orstructure as described above and shown in FIGS. 1 and 2. Designstructure 490 may then proceed to a stage 495 where, for example, designstructure 490: proceeds to tape-out, is released to manufacturing, isreleased to a mask house, is sent to another design house, is sent backto the customer, etc.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, element components,and/or groups thereof.

The corresponding structures, materials, acts, and equivalents of allmeans or step plus function elements in the claims below are intended toinclude any structure, material, or act for performing the function incombination with other claimed elements as specifically claimed. Thedescription of the present invention has been presented for purposes ofillustration and description, but is not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention. Theembodiment was chosen and described in order to best explain theprinciples of the invention and the practical application, and to enableothers of ordinary skill in the art to understand the invention forvarious embodiments with various modifications as are suited to theparticular use contemplated

The flow diagrams depicted herein are just one example. There may bemany variations to this diagram or the steps (or operations) describedtherein without departing from the spirit of the invention. Forinstance, the steps may be performed in a differing order or steps maybe added, deleted or modified. All of these variations are considered apart of the claimed invention.

While the exemplary embodiments to the invention have been described, itwill be understood that those skilled in the art, both now and in thefuture, may make various improvements and enhancements which fall withinthe scope of the claims which follow. These claims should be construedto maintain the proper protection for the invention first described.

1. A method for cutting a linear charged polymer inside a nanopore,comprising: applying a first voltage to create an electric field in afirst direction; applying a second voltage to create an electric fieldin a second direction, wherein the first direction is opposite to thesecond direction; and when the electric field in the first direction andthe electric field in the second direction are applied to a linearcharged polymer inside a nanopore, causing the linear charged polymer tobe cut at a location within a predetermined precision.
 2. The method ofclaim 1, further comprising applying a third voltage to be a biasingvoltage that moves the linear charged polymer inside the nanopore. 3.The method of claim 1, wherein the causing the linear charged polymer tobe cut at the location comprises stretching the linear charged polymeruntil the linear charged polymer breaks at the location within apredetermined precision.
 4. The method of claim 1, wherein when theelectric field in the first direction and the electric field in thesecond direction are applied to the linear charged polymer inside thenanopore and causes the linear charged polymer to be cut at thelocation, the linear charged polymer is cut into pieces at everypredetermined length.
 5. The method of claim 1, wherein when theelectric field in the first direction and the electric field in thesecond direction is applied to the linear charged polymer inside thenanopore and causes the linear charged polymer to be cut at thelocation, the linear charged polymer is cut at a predetermined distancebased on a particular pattern of monomers.
 6. The method of claim 1,wherein when the electric field in the first direction and the electricfield in the second direction are applied to the linear charged polymerinside the nanopore and causes the linear charged polymer to be cut atthe location, the linear charged polymer is cut between two monomers ofthe backbone of the linear charged polymer.
 7. The method of claim 1,wherein the linear charged polymer comprises at least one of a cationicpolysaccharide, deoxyribonucleic acid (DNA), and ribonucleic acid (RNA).8. The method of claim 1, wherein both the first voltage and the secondvoltage are applied such that the electric field in the first directionand the electric field in the second direction are strong enough tobreak the linear charged polymer at the precise location.
 9. The methodof claim 1, wherein when the electric field in the first direction andthe electric field in the second direction are applied to the linearcharged polymer inside the nanopore and causes the linear chargedpolymer to be cut at the precise location, the linear charged polymer iscut between consecutive charges of a backbone of the linear chargedpolymer.
 10. An apparatus for cutting a linear charged polymer inside ananopore, comprising: a reservoir filled with a conductive fluid; amembrane separating the reservoir, the membrane comprising electricalconductive layers and insulating layers; and a nanopore through themembrane; wherein a first voltage is applied to the conductive layers tocreate an electric field in a first direction; wherein a second voltageis applied to the conductive layers to create an electric field in asecond direction, the first direction being opposite to the seconddirection; and wherein when the electric field in the first directionand the electric field in the second direction are applied to a linearcharged polymer inside the nanopore, the electric field in the firstdirection and the electric field in the second direction cause thelinear charged polymer to be cut at a location.
 11. The apparatus ofclaim 10, wherein a third voltage is applied as a biasing voltage thatmoves the linear charged polymer inside the nanopore.
 12. The apparatusof claim 10, wherein to cause the linear charged polymer to be cut atthe location comprises stretching the linear charged polymer until thelinear charged polymer breaks at the location.
 13. The apparatus ofclaim 10, wherein when the electric field in the first direction and theelectric field in the second direction are applied to the linear chargedpolymer inside the nanopore and causes the linear charged polymer to becut at the location, the linear charged polymer is cut into pieces everypredetermined length.
 14. The apparatus of claim 10, wherein when theelectric field in the first direction and the electric field in thesecond direction are applied to the linear charged polymer inside thenanopore and causes the linear charged polymer to be cut at thelocation, the linear charged polymer is cut at a predetermined distanceaccording to a particular pattern of monomers.
 15. The apparatus ofclaim 10, wherein when the electric field in the first direction and theelectric field in the second direction are applied to the linear chargedpolymer inside the nanopore and causes the linear charged polymer to becut at the location, the linear charged polymer is cut between anelectrostatic cleavage of a backbone of the linear charged polymer. 16.The apparatus of claim 10, wherein the linear charged polymer comprisesat least one of a cationic polysaccharide, deoxyribonucleic acid (DNA),and ribonucleic acid (RNA).
 17. The apparatus of claim 10, wherein boththe first voltage and the second voltage are applied such that theelectric field in the first direction and the electric field in thesecond direction are strong enough to break the linear charged polymerat the location.
 18. The apparatus of claim 10, wherein when theelectric field in the first direction and the electric field in thesecond direction are applied to the linear charged polymer inside thenanopore and causes the linear charged polymer to be cut at thelocation, the linear charged polymer is cut between an electrostaticcleavage of a backbone of the linear charged polymer.
 19. A system forcutting a linear charged polymer inside a nanopore, comprising: anapparatus comprising: a reservoir filled with a conductive fluid; amembrane separating the reservoir, the membrane comprising electricallyconductive layers and electrically insulating layers; and a nanoporethrough the membrane; and a first voltage bias, wherein the firstvoltage bias is applied to the conductive layers to create an electricfield in a first direction; a second voltage bias, wherein the secondvoltage bias is applied to the conductive layers to create an electricfield in a second direction, the first direction being opposite to thesecond direction; and wherein when the electric field in the firstdirection and the electric field in the second direction are applied toa linear charged polymer inside the nanopore, the electric field in thefirst direction and the electric field in the second direction cause thelinear charged polymer to be cut at a location.
 20. The system of claim19, further comprising a library comprising linear charged polymerfragments that have been cut.
 21. The system of claim 20, furthercomprising a library of DNA fragments that have been cut.
 22. The systemof claim 21, wherein the library of DNA fragments is a proportion ofdifferent lengths.
 23. The system of claim 22, wherein the proportion ofdifferent lengths comprises a first length, a second length, through alength N; wherein the library comprises a predetermined number of thefirst length, a predetermined number of the second length, and apredetermined number of the length N; and wherein the length Nrepresents a last of the different lengths.